1. Field of the Invention
The present invention relates to the field of analytical chemistry, and particularly to the study of fluorescence and phosphoresence phenomena in the biochemical, biological and biophysical arts.
2. Discussion of the Prior Art
The use of fluorescence spectroscopy for the study of the dynamics of macromolecules is becoming more widespread as more sophisticated instrumentation is being developed. Although fluorescence spectroscopy has developed into a widely accepted technique in the physical and chemical sciences as well as in the biological sciences, the practical utility of fluorescence methods is still limited by the availability of fluorescence spectroscopy instrumentation capable of measuring such events accurately.
Fluorescence is the rapid decay from a higher to a lower state of the same multiplicity. The natural time window of fluorescence is suitable to resolve dynamic events occurring in the nanosecond (ns) to pico-second (ps) time region. The above characteristics, coupled with the sensitivity of the excited state of a fluorophore to the physicochemical properties of its environment, is a major reason why fluorescence spectroscopy techniques are so frequently used in the study of micro-biological structures and functions.
The greatest interest is in measuring dynamic events displayed in the kinetics of intensity decay (fluorescence lifetimes) and anisotropy decay. The fluorescence lifetime reflects not only the intrinsic radiative rate of the excited state, but also the interactions of the fluorophore with the environment. Anisotropy decay measures the displacement of the emission transition dipole with time after excitation and thus reflects the rotational motion of the fluorophore. The rate and the amplitude of the rotational motion in a given time are themselves dependent on the free volume, the microscopic viscosity of the local environment and the forces acting on the excited molecule. Therefore, anisotropy decay indirectly describes the structure and dynamics of the fluorophore's environment. Clearly, a detailed study of the fundamental fluorescence observables (spectrum, quantum yield, lifetime and anisotropy) can provide substantial information about a biological macromolecule and its surrounding. Additional insight can be gained, if the system is physically or chemically perturbed, for example, by temperature or viscosity change or the presence of fluorescence quenching agents. The frequently complex fluorescence signal from biological systems does not easily yield to mathematical analysis and it may be difficult to correlate a physical event with the result of the analysis.
The time decay of fluorescence is usually measured using one of two accepted, but different approaches. Measurements of fluorescence decay can be made in the time domain using the popular technique of correlated single photon counting (SPC), or in the frequency domain by determining the phase delay and the relative modulation of the fluorescence signal with respect to the exciting light. The modern study of fluorescence properties started with time domain fluorometry and has evolved into methods using frequency domain fluorometry. In the frequency domain, the frequency axis is examined one point at a time, while in the time domain, the full decay is collected at once; however, the collection of information in the time domain takes from several minutes to several hours depending upon the excitation source, while in the frequency domain, the data collection at a single frequency takes only a few seconds. Therefore, it is possible in the frequency domain to acquire an equivalent amount of information in a similar amount of time. Indeed, a great advantage in the frequency domain can be achieved if all frequencies can be collected at the same time.
The maximum time resolution of sequential multifrequency phase fluorometers is about 1 or 2 picoseconds, which compares favorably with time correlated single photon counting instruments. The decomposition of the decay curve using a sum of exponentials, may also be obtained from a multifrequency measurement applying a non-linear least squares routine. The analysis of a double and triple exponential decay may be performed on dedicated micro-computers.
Resolution of emission anisotropy decay is obtained by a measurement of the differential phase and modulation ratio of the horizontally and vertically polarized emission components, arising from vertically polarized excitation. This technique, originally developed for single modulation frequency operation, has become extremely powerful when coupled with a multifrequency phase fluorometer. Fast rotational correlation times on the order of 10 picoseconds and longer can be measured. Resolution of anisotropic rotational motions can also be obtained from a multifrequency data set using a non-linear least squares analysis. Restricted rotational motions can also be analyzed. The ability to perform direct differential measurement, such as the phase delay between the perpendicular and the parallel polarized components of the emissions, is a unique intrinsic characteristic of phase fluorometry and results in an improved time resolution.
Phase fluorometry has the intrinsic capability to perform phase sensitive detection, which provides a simple and powerful method to separate spectral components in a mixture of fluorophores. This separation is based on the principle that each emitting species in the mixture has a characteristic phase delay. The spectra of the overlapping components can be obtained with a single scan using our new approach of phase and modulation resolved spectra. This simple approach requires no fitting of the data. The resolution is instead obtained directly from the values of the phase and modulation.
The prior art shows a number of examples of systems utilizing frequency domain fluorometry techniques. The 1984 article "The Measurement and Analysis of Heterogeneous Emissions by Multi-frequency Phase and Modulation Fluorometry" by Jameson, Gratton, and Hall, Applied Spectroscopy Reviews, 20(1), pages 55-106 (1984) discloses two methods of multi-frequency phase and modulation fluorometry as well as a commercially available fluorometer. In addition, the article discloses a fluorometer the authors developed for research purposes. The commercially developed fluorometer, developed by SLM AMINCO, utilizes a xenon arc lamp to provide an excitation signal to generate the fluorescence emissions. The light supplied by the arc lamp is intensity modulated before impinging upon a sample to be studied. The light emitted by the (study) sample is detected by a photomultiplier, the second or third dynode of which is modulated at a frequency equal to the light modulation frequency plus a small additional frequency. This procedure is a cross-correlation technique, wherein the phase and modulation information of the emitted signal is transposed to a much lower frequency range where it can be interrogated. The phase delay and demodulation of the emitted signal relative to the scattered light is then calculated. The research fluorometer described in the article is a variable frequency cross-correlation phase fluorometer which utilizes an argon ion laser to provide an excitation beam to excite the fluorescence action and to provide a reference signal. The light supplied by the laser is sinusoidally modulated, and split into two beams, one signal is used to excite the study sample and the second signal is used as the reference signal. The reference signal and the signal emitted by the study sample are then passed through two photomultipliers wherein the cross-correlation processing described above is done. The outputs from both photomultipliers are then passed through identical sections of analog circuitry wherein the data is sequentially processed and displayed.
The 1986 article "A Multi-Frequency Phase Fluorometer using the Harmonic Content of a Mode Locked Laser" by Alcala and Gratton, Analytical Instrumentation, 14(3 and 4), pages 225-250 (1985) discloses a cross-correlation phase and modulation fluorometer which utilizes the harmonic content of a high repetition rate, mode locked laser. In the frequency domain a pulsed source provides a large series of equally spaced harmonic frequencies. The pulses from the laser are amplitude modulated and frequency doubled. The signal is then split into a reference beam and an excitation beam. The reference beam is directed to a first photomultiplier and the excitation beam is directed to a study sample and then the emission from the sample is detected by a second photomultiplier. The photomultipliers provide cross-correlated mixing which in addition to frequency translation also allows transfer of the phase and modulation information desired at the individual harmonic frequencies. The outputs from the photomultipliers are then passed through various forms of analog filtering circuits and amplifiers wherein the necessary phase and modulated data is sequentially derived from the outputs of the photomultipliers.
Frequency domain fluorometry in certain instances has the advantage of the rapid determination of single or double exponential fluorescence lifetimes which can be obtained by measurements at only one or two frequencies. This is not possible for systems where complex fluorescence decays must be resolved. In order to handle complex decays, a large number of modulation frequencies is needed to obtain the full decay information. The above disclosed fluorometers provide this capability only to a limited extent.
The above referenced articles disclose fluorometers that use frequency domain techniques as opposed to time domain techniques. Frequency domain fluorometers have the advantage of high accuracy and rapid determination of fluorescence lifetimes. However, the above referenced fluorometers utilize analog signal processing techniques after data collection. Unwanted effects on the signals of interest are caused by the bandwidth and non-linearity of the analog filters used in the above referenced fluorometers. In the analog electronics of most commercial frequency domain fluorometers, six pole active filters are utilized to perform the necessary filtering functions. These filters are hard to tune to the appropriate frequency, they suffer from thermal and drifting problems and have undesirable phase shift. The accuracy of lifetime measurement is limited by the analog signal processing portion of the fluorometers.